Pourable feed additives containing D-pantothenic acid and/or salts thereof, and process for their preparation

ABSTRACT

A composition including at least one of D-pantothenic acid and a salt of D-panthothenic acid, greater than 50 percent of non-biomass constituents of a D-pantothenic fermentation liquor, and 0 to 100% of the biomass from D-pantothenic fermentation liquor wherein composition is a solid, pourable composition in the form of particles.

This application is a continuation of application Ser. No. 09/895,318filed on Jul. 2, 2001, now U.S. Pat. No. 6,368,644.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to pourable animal feed additives based onfermentation liquor and containing D-pantothenic acid and/or a saltthereof, and to a process for the preparation of such additives.

2. Discussion of the Background

Pantothenic acid is produced worldwide on a scale of several thousandtons per year, and demand for the product is increasing. Much of thepantothenic acid that is produced is used for feeding productivelivestock such as poultry and pigs.

Pantothenic acid can be produced by biotechnology or by chemicalsynthesis. Biotechnology methods involve fermentation of suitablemicroorganisms in appropriate nutrient solutions. In chemical synthesis,DL-pantolactone is an important precursor. It is prepared in amulti-step process from formaldehyde, isobutylaldehyde and cyanide wherethe racemic mixture is resolved, the D-pantolactone is condensed withβ-alanine, and D-pantothenic acid is thus obtained.

The typical commercial form of pantothenic acid is the calcium salt ofD-pantothenic acid. The calcium salt of the racemic mixture ofD,L-pantothenic acid is also common.

The advantage of preparing pantothenic acid by means of fermentation ofmicroorganisms is that the desired stereoisomeric form, namely the Dform, which is free of L-pantothenic acid, is formed directly.

Various types of bacteria, such as, for example, Escherichia coli,Arthrobacter ureafaciens, Corynebacterium erythrogenes, Brevibacteriumammoniagenes, and also yeasts, such as, for example, Debaromycescastellii, are able to produce D-pantothenic acid under suitablefermentation conditions, as is shown in EP-A-0 493 060, EP-A-0 590 857and WO 97/10340. Especially suitable microorganisms are the Escherichiacoli IFO3547 derivatives described therein, such as, for example, thestrains FV5069/pFV31 or FV5069/pFV202.

In the preparation of D-pantothenic acid by fermentation, as isdescribed in EP-A-0 493 060, EP-A-0 590 857 and WO 97/10340, amicroorganism capable of producing D-pantothenic acid is cultivated in asuitable nutrient medium and the D-pantothenic acid that forms is thenisolated, purified and prepared in the form of the calcium salt in acomplicated manner.

Suitable nutrient media contain a carbon source, a nitrogen source, aphosphorous source, salts, trace elements and vitamins, and optionally,complex media additives, such as yeast extract. Examples of the carbonsource include glucose, starch flour hydrolysate, sucrose and molasses.An example of the nitrogen source is ammonium sulfate. An example of thephosphorous source is potassium phosphate.

According to the current prior art described in WO96/33283 and EP-A-0590857, the calcium salt of D-pantothenic acid is obtained from thepantothenic acid-containing fermentation liquor by means of a complexisolation and purification operation. After first separating off thebiomass by filtration or centrifugation, the filtrate is furtherpurified by means of activated carbon or by column chromatography. Afterreaction of the resulting solutions with calcium hydroxide, the desiredCa salt is allowed to crystallize.

According to WO 96/33283, the filtrate is decoloured with activatedcarbon in the first column. A pH value of 3.0 is adjusted usingconcentrated hydrochloric acid, and the liquid is then purifiedcontinuously over two further columns packed with activated carbon.

Elution of the D-pantothenic acid takes place with the aid of methylalcohol. Subsequent neutralisation using Ca(OH)₂ powder yields asolution from which calcium D-pantothenate is obtained bycrystallisation at 5° C.

In the method described in EP-A-0 590 857, the filtrate is firstpurified with the aid of cation- and anion-exchanger columns. Elutiontakes place with hydrochloric acid. The eluted fraction is thenneutralised using Ca(OH)₂; activated carbon is added thereto and thewhole is filtered off. The filtrate that is obtained is then extractedin a low molecular weight alcohol (methanol, ethanol, isopropanol), andcalcium D-pantothenate is obtained by crystallisation.

The calcium D-pantothenate prepared in the described manner is used asan additive in animal feed.

OBJECT OF THE INVENTION

According to the prior art, salts of D-pantothenic acid orD,L-pantothenic acid are obtained by chemical synthesis or fromfermentation liquors and then added in pure form to feeds.

The object of the invention is to provide more readily processable formsof D-pantothenic acid and its salts and processes for the preparationthereof for feeds.

DESCRIPTION OF THE INVENTION

The present invention provides a pourable animal feed additive based onfermentation liquor and containing D-pantothenic acid and/or saltsthereof. The feed additive is characterized in that

a) it contains the biomass formed during the fermentation in an amountof from ≧0 to 100%; and

b) it contains at least the predominant part of the further constituentsof the fermentation liquor; and

c) it is in solid form, in a particle size distribution of from 20 to2000 μm, especially from 50 to 800 μm, more especially from 150 to 600μm, and is pourable.

In a preferred embodiment of the invention, the pourable animal feedadditive containing D-pantothenic acid and/or salts thereof ischaracterized in that it additionally contains, in solid form, an amountof chloride-containing constituents in a concentration of <3 mg per g ofadditive, preferably <2 mg per g of additive and especially <1.5 mg perg of additive.

The additives are generally compacted, granulated, or fine-grained, butin any case, pourable form, according to requirements, and containvarying amounts of biomass. The apparent density is from 200 to 800kg/m³, especially approximately from 400 to 700 kg/m³.

Pourable in the context of this invention means freely flowingnon-clumped particles of a predetermined size which are able to bedispensed from a container. The additives are readily pourable andstorage stable.

If the biomass is separated off further, inorganic solids, for examplethose which have been added during the fermentation, are generallyremoved. In addition, the additive according to the invention containsat least the predominant part of the further substances, especiallyorganic, that have been formed or added and are dissolved in thefermentation liquor, insofar as they have not been separated off bysuitable processes.

Such substances include organic by-products that are produced andsecreted in addition to D-pantothenic acid by the microorganisms used inthe fermentation. They include L-amino acids, selected from the groupL-methionine, L-lysine, L-valine, L-threonine, L-alanine orL-tryptophan, especially L-valine. They also include organic acidscarrying from one to three carboxyl groups, such as, for example, aceticacid, lactic acid, citric acid, malic acid or fumaric acid. Finally,they also include sugars, such as, for example, trehalose. Suchcompounds may be desirable, in that they improve the nutritional valueof the additive.

In a preferred form there is prepared a fermentation liquor containingD-pantothenic acid and/or salts thereof, wherein

a) the fermentation takes place in a substantially chloride-free medium,

b) the resulting fermentation liquor, optionally after separation of thebiomass and concentration, is dried, compacted, spray dried, spraygranulated or granulated or applied to a carrier or embedded in astabilising matrix.

The fermentation liquors which are suitable for the process according tothe invention are obtained using microorganisms suitable for theproduction of D-pantothenic acid and that contain D-pantothenic acidand/or salts thereof. The salts are generally sodium, potassium,ammonium, magnesium or calcium salt.

The microorganisms may be fungi or yeasts, such as, for example,Debaromyces castellii, or Gram-positive bacteria, for example of thegenus Corynebacterium, or Gram-negative bacteria, such as, for example,those of the family Enterobacteriaceae. In the case of the family of theEnterobacteriaceae, special mention may be made of the genus Escherichiaand of the species Escherichia coli. Within the species Escherichiacoli, mention may be made of the so-called K-12 strains, such as, forexample, strains MG1655 or W3110 (Neidhard et al.: Escherichia coli andSalmonella. Cellular and Molecular Biology (ASM Press, WashingtonD.C.)), or of the Escherichia coli wild type strain IFO3547 (Institutfür Fermentation, Osaka, Japan) and mutants derived therefrom both ofwhich are incorporated herein by reference. Of the strains produced fromIFO3547, FV5069/pFV31 (EP-A-0 590 857) and FV5069/pFV202 (WO 97/10340)are prominent. In the case of the genus Corynebacterium, special mentionmay be made of the species Corynebacterium glutamicum.

The above-described microorganisms can be cultivated for the purposes ofD-pantothenic acid production continuously or discontinuously by thebatch or fed batch or repeated fed batch process. A summary of knowncultivation methods is described in the textbook by Chmiel(Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik (GustavFischer Verlag, Stuttgart, 1991) or in the textbook by Storhas(Bioreaktoren und periphere Einrichtungen (Vieweg Verlag,Braunschweig/Wiesbaden, 1994) both of which are incorporated herein byreference.

The culture medium to be used must meet the requirements of themicroorganisms to be used in a suitable manner. The fermentation mediumis substantially free of chloride-containing constituents. According tothe invention, the concentration of chloride ions in the productionfermenter is <300 mg/l, preferably <200 mg/l and very especiallypreferably <150 mg/l. There may be used as the carbon source sugars andcarbohydrates such as glucose, saccharose, lactose, fructose, maltose,molasses, starch and cellulose, oils and fats, such as soybean oil,sunflower oil, groundnut oil and coconut oil, fatty acids such aspalmitic acid, stearic acid and linoleic acid, alcohols such as glyceroland ethanol, and organic acids such as acetic acid. Those substances maybe used individually or in the form of a mixture. There may be used asthe nitrogen source organic nitrogen-containing compounds, such aspeptones, yeast extract, meat extract, malt extract, corn steep liquor,soybean flour and urea, or inorganic compounds, such as ammoniumsulfate, ammonium phosphate, ammonium carbonate and arumonium nitrate.The nitrogen sources may be used individually or in the form of amixture. There may be used as the phosphorus source potassium dihydrogenphosphate or dipotassium hydrogen phosphate or the correspondingsodium-containing salts. The culture medium must also contain salts ofmetals such as magnesium sulfate or iron sulfate, which are necessaryfor growth. Finally, essential growth substances, such as amino acidsand vitamins, may be used in addition to the above-mentioned substances.Precursors of D-pantothenic acid, such as aspartate, β-alanine,ketoisovalerate, ketopantoic acid or pantoic acid, and, optionally,salts thereof, may also be added to the culture medium. The mentionedsubstances may be added to the culture in the form of a single batch, orthey may be fed in a suitable manner during the cultivation.

In order to control the pH value, ammonia or ammonia water or otherbasic compounds, such as sodium hydroxide, potassium hydroxide orcalcium hydroxide are used. If acid compounds are required to controlthe pH value, phosphoric acid or sulfuric acid may expediently be used.In order to prepare the calcium salt of pantothenic acid directly,calcium hydroxide in the form of an aqueous suspension is used duringthe fermentation. In order to control the development of foam,anti-foams such as fatty acid polyglycol esters, may be used. In orderto maintain the stability of plasmids, suitable substances having aselective action, for example antibiotics, are optionally added to themedium. In order to maintain aerobic conditions, oxygen or gas mixturescontaining oxygen such as air, are introduced into the culture. Thetemperature of the culture is normally from 20° C. to 45° C. andpreferably from 25 ° C. to 40° C. The culture is continued until themaximum amount of D-pantothenic acid has formed, which is generallywithin a period of 10 hours to 160 hours.

The fermentation liquors thus obtained usually have a content of drymatter of 7.5 to 25 wt. % and contain from 2 to 20 wt. % D-pantothenicacid. Fermentation processes in which D-pantothenic acid is present inthe dry matter in an amount of at least 20 wt. % when the fermentationis complete are especially advantageous. It is also advantageous for theamount of sugar in the fermentation to be limited at least at the end ofthe fermentation, but advantageously for at least 30% of the duration ofthe fermentation. That means that the concentration of usable sugar inthe fermentation medium is maintained at, or reduced to from ≧0 to 3 g/lduring that time.

For the preparation of the additives according to the invention, thefermentation liquors containing D-pantothenic acid and/or salts thereofare preferably first freed of all or part of the biomass by knownseparation methods such as centrifugation, filtration, decantation or acombination thereof. However, it is also possible according to theinvention to leave all of the biomass in the fermentation liquor. Thesuspension obtained in that manner is then concentrated preferably tonot more than 60 wt. % dry matter and worked up to a powder, for examplewith the aid of a spray drier or a lyophilising apparatus. The powder isthen converted into a coarser-grained, readily pourable, storable andlargely dust-free product by suitable compacting or granulatingprocesses. In the granulating or compacting operation it is advantageousto use conventional organic or inorganic auxiliary substances, orcarriers, such as starch, gelatin, cellulose derivatives or similarsubstances, as are conventionally employed as binders, gelling agents orthickeners in the processing of foodstuffs or feeds, or furthersubstances such as, for example, silicas, silicates or stearates.

Alternatively, the product may be applied to an organic or inorganiccarrier substance that is known and conventionally employed in theprocessing of feeds, such as, for example, silicas, silicates, meals,brans, flours, starches, sugars or the like, and stabilised by means ofconventional thickeners or binders. Relevant examples and processes aredescribed in the literature (Die Mühle+Mischfuttertechnik 132 (1995) 49,page 817) which is incorporated herein by reference.

The novel solid products according to the invention that containD-pantothenic acid and/or salts thereof and that can be prepared by theabove-described process contain from 20 to 80 wt. %, preferably from 30to 75 wt. %, D-pantothenic acid. They generally contain inorganicconstituents in an amount of from 2.5 to 25 wt. % and optionally organicby-products in an amount of from >0 to 30 wt. %. The content of drybiomass is from ≧0 to 35 wt. %. The water content is preferably <5 wt.%.

The novel products according to the invention that contain D-pantothenicacid and/or salts thereof and that are prepared by the above-describedprocess are distinguished by a particle size distribution of from 20 μmto 2000 μm, preferably from 50 μm to 800 μm and especially from 150 μmto 600 μm. The content of very fine dust (<10 μm) is approximately from0 wt. % to 10 wt. %, preferably approximately from 0 wt. % to 5 wt. %.The product is used as a feed additive.

The concentration of D-pantothenic acid can be determined by knownmethods (Velisek; Chromatographic Science 60, 515-560 (1992)).

The particle size distribution can be determined by methods of laserdiffraction spectrometry. Corresponding methods are described in thetextbook on “Teilchengrössenmessung in der Laborpraxis” by R. H. Müllerand R. Schuhmann, Wissenschaftliche Verlagsgesellschaft Stuttgart (1996)or in the textbook “Introduction to Particle Technology” by M. Rhodes,Verlag Wiley & Sons (1998) both of which are incorporated herein byreference.

EXAMPLES

The present invention is explained in greater detail below by means ofExamples. To that end, tests have been carried out using theD-pantothenic acid-producing strain Escherichia coli 5069/pFV31, whichhas been deposited as FERM-BP 4395 in accordance with the BudapestTreaty at the Fermentation Research Institute, Agency of IndustrialScience and Technology in 1-1-3, Higashi, Tsukuba-shi, Ibaraki (Japan)(EP-A-0590857).

The measurements were carried out on a Cilas 920 laser diffractionspectrometer from Quanto Chrome (Odelzhausen, Germany). Evaluation ofthe measurement results was carried out as specified in GermanIndustrial Standard DIN 66141 for representation of the particle sizedistribution.

Example 1

Preparation of the Calcium Salt of D-pantothenic Acid in a FermentationLiquor

1. Preparation of Inoculum (Master Cell Bank)

A sample of Escherichia coli FV5069/pFV31 was spread on LBG agar towhich 50 μg per ml ampicillin had been added. The agar plate culture wasincubated for 17 hours at 37° C. and then stored in a refrigerator at+4° C. Selected individual colonies were then multiplied further in LBGbouillon. LBG bouillon has the following composition: 10 g/l peptone, 5g/l yeast extract, 5 g/l NaCl and 1 g/l glucose. LBG agar additionallycontains 12 g/l agar. Ready-made preparations can be obtained fromGibco/BRL (Paisley, Scotland, Great Britain) as LB broth base or LBagar. After the addition of 1 g/l glucose, the indicated media are thenobtained. 10 ml cultures, which were contained in 100 ml Erlenmeyerflasks, were incubated for 16 hours at 37° C. and 180 rpm in an ESRincubator from Kühner AG (Birsfelden, Switzerland). The cell suspensionwas then separated by centrifuging in a J-6B centrifuge from Beckmann(Hanover, Germany) for 15 minutes at 4000 rpm. The cell pellet wasre-suspended in 10 ml of LBG medium to which 20% glycerol had beenadded; 1 ml was introduced under sterile conditions into each of 10aliquots and frozen at −70° C. Those cultures were used as the mastercell bank.

For the preparation of a working cell bank, LBG medium to which 50 μg/mlampicillin had been added was divided into 10 ml portions in 100 mlErlenmeyer flasks and then inoculated with 100 μl of the above-describedmaster cell bank. Incubation was carried out for 16 hours at 37° C. and180 rpm in an ESR incubator from Kühner AG (Birsfelden, Switzerland).

After incubation, the optical density (OD) of the culture suspension wasdetermined at a measuring wavelength of 660 nm using an LP2W photometerfrom Dr. Lange (Berlin, Germany). It was 3.5. The cell suspension wasthen introduced under sterile conditions into sterile 30 ml polyethylenetubes from Greiner (Frickenhausen, Germany) and separated bycentrifuging at 2500 rpm for 15 minutes using a J-6B centrifuge fromBeckmann (Hanover, Germany). The biomass that had been separated off wasre-suspended in 10 ml of LBG medium to which 20% glycerol had beenadded. The cell suspension was then introduced in 500 μl portions, understerile conditions, into 1 ml sterile tubes from Nalgene (New York,U.S.A.) and frozen at −70° C. The preserved batches so prepared wereused as the working cell bank.

2. Preparation of a Fermentation Liquor Containing CalciumD-pantothenate

For the preparation of a fermentation liquor containing calciumD-pantothenate, the working cell bank was first multiplied in a shakingflask culture, and the latter was used to inoculate a pre-fermenter. Thepre-fermenter culture was used to inoculate the production fermenter.

SKA medium (Table 1) was used for the shaking flask culture. SKA mediumwas prepared as follows: 7.0 g of (NH₄)₂SO₄, 0.5 g of KH₂PO₄, 1.0 g ofK₂HPO₄, 0.5 g of MgSO₄.7H₂O, 0.01 g of MnSO₄.H₂O, 0.001 g of ZnSO₄.7H₂O,0.005 g of Fe₂(SO₄)₃ and 20 g of corn steep liquor, which had previouslybeen adjusted to pH 6.8 using 25% ammonia solution, were weighed into a1 liter glass beaker and then made up to 825 g with distilled water.That salt solution containing corn steep liquor was sterilised in anautoclave at 121° C. for 20 minutes. In addition, a solution consistingof 25 g of glucose and 0.002 g of thiamine.HCl was made up to 125 g withdistilled water and sterilised by filtration. 10 g of CaCO₃ were weighedinto a 100 ml flask and sterilised in an autoclave at 123° C. for 20minutes. SKA medium was obtained by combining the above-mentioned twocomponents with the salt solution containing corn steep liquor.

The SKA medium was divided into 12.5 ml portions in 100 ml Erlenmeyerflasks and was then inoculated with 0.5 ml of a cell suspension. Thecell suspension used was a preserved batch of the working cell culturediluted 1:100 with sterile physiological saline. Incubation was carriedout for 20 hours at 32° C. and 150 rpm on an RC-1-TK incubator fromInfors AG (Bottmingen, Switzerland). The optical density determinedthereafter at a measuring wavelength of 660 nm (OD 660) was 12.5.

For the inoculation of 20 kg of A1-102 pre-culture medium, which wascontained in a 42 liter stirred reactor fermenter from Bioengineering(Wald, Switzerland, model LP-42), 0.5 ml of SKA medium was diluted 1:100and 50 ml of that suspension were added to the fermenter. Thepre-culture medium A1-102 contained the constituents listed in Table 2.The culture was cultivated for 15.5 hours at a temperature of 37° C.,with volume-specific aeration of 0.5 volume/volume/minute (vvm), at anoxygen partial pressure of 20% of air saturation and at a pH of 6.5,until an OD660 of 11.3 was reached.

For the inoculation of 5830 g of M1-380 main culture medium, which wascontained in 14 liter stirred reactor fermenters from B. Braun (BBI,Germany, Melsungen, model Biostat E/ED), 423 ml of the secondpre-culture in A1-102 medium were added. M1-380 main culture mediumcontained the constituents listed in Table 3. The culture was firstcultivated for 6.5 hours at a temperature of 37° C., withvolume-specific aeration of 0.75 vvm, with minimum stirring of 400 rpmand at a pH of 6.5, until an OD660 of 18.6 and an oxygen partialpressure of 2% of air saturation had been reached. The culture was thencultivated for a further 41 hours at a temperature of 37° C., an oxygenpartial pressure of 2% of air saturation and a pH value of 6.0, until anOD660 of 66.8 had been reached. After a fermentation time of 13 hours,β-alanine was fed in in a concentration of 152.7 g in 570 ml of H₂O overa period of 34.5 hours. After a fermentation time of 21.5 hours, a tenpercent Ca(OH)₂ solution was metered in over a period of 26 hours inorder to control the pH. 3.43 kg of M2-257 medium having a glucoseconcentration of 650.8 g/l and a concentration of thiamin.HCl of 35.7g/l were fed in in the course of 41 hours.

The optical density (OD) was then determined at a measuring wavelengthof 660 nm using an LP1W type digital photometer from Dr. Bruno LangeGmbH (Berlin, Germany), and the concentration of D-pantothenic acid thathad formed was determined by means of HPLC (Hypersil APS 2 5 μm, 250×5mm, RI detection).

A calcium D-pantothenate concentration of 49.7 g/l, measured asD-pantothenic acid, was determined in the final fermentation sampleafter 70.0 hours.

The content of D-pantothenic acid was determined with the aid of a typeM321 HPLC (high-performance liquid chromatography) apparatus from Knauer(Berlin, Germany) by means of RI (refractive index) detection using aHypersil APS2 amino phase of particle size 5 μm.

TABLE 1 Composition of SKA medium Concentration Component (per liter)Glucose 25.0 g Corn steep liquor 20.0 g (NH₄)₂SO₄ 7.0 g KH₂PO₄ 0.5 gK₂HPO₄ 1.0 g MgSO₄.7H₂O 0.5 g FeSO₄.7H₂O 5 mg MnSO₄.H₂O 10 mg ZnSO₄.7H₂O1 mg CaCO₃ 10 g Thiamin chloride.HCl 2 mg Structol 0.7 g

TABLE 2 Composition of A1-102 medium Concentration Component (per liter)Glucose 25.0 g Corn steep liquor 20.0 g (NH₄)₂SO₄ 7.0 g KH₂PO₄ 0.5 gK₂HPO₄ 1.0 g MgSO₄.7H₂O 0.5 g FeSO₄.7H₂O 10 mg MnSO₄.H₂O 10 mg Thiaminchloride.HCl 3 mg Structol 0.6 g

TABLE 3 Composition of M1-1380 medium Concentration Component (perliter) Glucose 18.0 g Corn steep liquor 40.0 g β-Alanine 15.0 g(NH₄)₂SO₄ 11.8 g KH₂PO₄ 0.6 g K₂HPO₄ 1.2 g MgSO₄.7H₂O 0.67 g MnSO₄.H₂O10 mg Thiamin chloride.HCl 1.6 mg Structol 0.6 g

Example 2

Preparation of Finely Divided Calcium D-pantothenate from FermentationLiquor

The biomass was first separated from a calcium D-pantothenicacid-containing fermentation liquor that had been prepared according tothe process of Example 1 and contained approximately 4.9 wt. %D-pantothenic acid. To that end, 90 liters of the above-mentionedfermentation liquor were filtered off by cross-flow filtration using a0.22 μm microfiltration membrane in a CERAFLO MSP005756 filtrationapparatus from Millipore (Bad Homburg, Germany).

The liquor so treated was then concentrated to a liquid content ofapproximately 30% dry content in a Rotavapor R-152 rotary evaporatorfrom Büchi, Switzerland at from 40 to 80° C., in vacuo. The liquor soconcentrated was then spray dried in order to prepare the calcium saltof D-pantothenic acid. To that end there was used a Technikum spraydrier from Niro (Copenhagen, Denmark) of the NIRO Minor type having anatomising plate (120 mm diameter; speed of rotation 135 m/sec.) at aninlet temperature of 175° C., an outlet temperature of 80° C. and adrying gas throughput of 525 m³/hour. For the purposes of betterdischarge of the product, Sipernat 22 from Degussa-Hüls AG (Frankfurt amMain, Germany) was added as powder auxiliary to the drying gas stream ina ratio of 5 wt. %, based on dry matter in the concentrate.

The calcium D-pantothenate-containing product so prepared had aD-pantothenic acid content of 48.5 wt. %, was pourable and had anapparent density of 460 kg/m³ with a mean particle size of 34 μm.

TABLE 4 Designation Diameter Content [%] Finedust <10 μm 10 Dust 10-20μm 20 Powder/dust 20-50 μm 50 Powder >50 μm 20

Example 3

Preparation of D-calcium Pantothenate Having a Particle Size >100 μm byCompaction in a Roll Compactor and Sieving

A calcium D-pantothenic acid-containing dust-like product, which hadbeen prepared according to the process of Example 2 and containedapproximately 48.5 wt. % D-pantothenic acid and had a mean particle sizeof 34 μm, was compacted by means of a roll compactor having cigar-likerolls (Pharmapaktor from BEPEX of type L200/50 P) with a compressiveforce of from 40 to 90 Newtons. The speed of rotation of the rolls was10 revolutions per minute. The compacted product so prepared was thenbroken down to a particle size distribution of from 200 to 400 μm incomminution sieves. The yield in the individual particle fractions issummarised in Table 5.

The compacted product was distinguished by a markedly lower content offine dust and substantially improved flow behaviour as compared with thepulverulent starting product.

TABLE 5 Designation Diameter Content [%] Fine dust <10 μm  5 Dust 10-50μm  5 Fines 50-200 μm 20 Mean particle size 200-400 μm 50 Oversizeparticles >400 μm 20

The fraction “mean particle size” was isolated by sieving and representsthe product. The product so prepared had a content of approximately 40.7wt. %, measured as D-pantothenic acid, and had an apparent density of630 kg/m³.

Example 4

Preparation of D-calcium Pantothenate Having a Mean Particle Size offrom 200 to 400 μm by Build-up Granulation in a Fluidised-bed Granulator

4.1 Use of Water as the Granulation Binder

Pulverulent product containing calcium D-pantothenic acid, which hadbeen prepared from a fermentation solution containing calciumD-pantothenic acid by spray drying according to the process described inExanple 2, was subsequently processed further in a fluidised-bedgranulator by spraying with a particular amount of water.

To that end, 300 g of the dust-like calcium D-pantothenicacid-containing product prepared according to Example 2 were placed in alaboratory fluidised-bed apparatus from Aeromatics Niro (Copenhagen,Denmark). At a fluidised bed temperature of 50° C. and a waste gastemperature of 30° C., 3 g of water per minute were sprayed in via ametering device. The fluidised gas temperature was from 70 to 80° C. Theparticle size distribution of the product so prepared is shown in Table6.

TABLE 6 Designation Diameter Content [%] Fine dust <10 μm 1 Dust 10-50μm 4 Fines 50-200 μm 20  Mean particle size 200-400 μm 75  Oversizeparticles >400 μm 0

The content, measured as D-pantothenic acid, was determined as 38.1 wt.%. The product was almost dust-free. The apparent density was 310 kg/m³.The product was very readily pourable.

4.2 Use of a Concentrate Containing Calcium Pantothenate as theGranulation Binder

A pulverulent product containing calcium D-pantothenic acid, which hadbeen prepared from a calcium D-pantothenic acid-containing fermentationsolution by spray drying according to the process described in Example2, was processed further in another test in a fluidised-bed granulatorby spraying with a particular amount of concentrated calciumD-pantothenate solution having a content of dry matter of approximately50 wt. %.

To that end, 1000 g ofthe dust-like calcium D-pantothenicacid-containing product prepared according to the process described inExample 2 were placed in a laboratory fluidised-bed apparatus, whichoperated batchwise, from Glatt (Binzen, Germany). At a fluidised bedtemperature of approximately from 40 to 45 ° C. and an inlet airtemperature of approximately 80° C., approximately 5 g of theabove-described concentrate per minute were sprayed into the laboratoryfluidised-bed apparatus. The particle size distribution of the productso prepared is shown in Table 7.

TABLE 7 Designation Diameter Content [%] Fine dust <10 μm 1 Dust 10-50μm 2 Fines 50-200 μm 18  Mean particle size 200-400 μm 79  Oversizeparticles >400 μm 0

The content, measured as D-pantothenic acid, was determined as 38.9 wt.%. The product was almost dust-free. The apparent density was 400 kg/m³.

By means of the process described herein, or by means of a differentspray, fluidised-bed, stirring or mixing process, the concentratecontaining D-calcium pantothenate or D-pantothenic acid is sprayed ontoother conventional organic or inorganic carriers or auxiliarysubstances, such as silicas, silicates, meals, brans, flours, starches,sugars or the like, and granulated, optionally with the use of binders,gelling agents or other formulation auxiliaries.

Example 5

Preparation of D-calcium Pantothenate Having a Mean Particle Size from100 to 400 μm by Mixing and Granulation in a Vacuum Drier

The biomass was first separated from a calcium D-pantothenicacid-containing fermentation liquor that had been prepared according tothe process of Example 1 and contained approximately 4.9 wt. %D-pantothenic acid. To that end, 90 liters of the above-mentionedfermentation liquor were filtered off by cross-flow filtration using a0.22 μm microfiltration membrane in a CERAFLO MSP005756 filtrationapparatus from Millipore (Bad Homburg, Germany).

The liquor so treated was then concentrated to a liquid content ofapproximately 50 wt. % dry content in a Rotavapor R-152 rotaryevaporator from Büchi, Switzerland, at from 40 to 80° C., in vacuo. Thecalcium D-pantothenic acid-containing concentrate so prepared having aD-pantothenic acid content of 28.8 wt. % was then mixed with a silica(Sipernat 22, Degussa-Hüls AG, Frankfurt Germany) with the aid of avacuum drier (type VT 130, Gebrüder Lödige Maschinenbau GmbH, Paderbom,Germany) to form a freely flowable granulate. 15 kg of the silica(Sipernat 22, Degussa-Hüls AG, Germany) were first placed in the vacuumdrier (type VT 130, Gebrüder Lödige Maschinenbau GmbH, Germany), andthen 39.0 kg of the calcium D-pantothenic acid-containing concentrateprepared above were added at a rate of 2.0 kg per minute under a vacuumof 200 mbar, with the material to be mixed having a temperature of 45 °C., and with a stirrer performance of 120 rpm (revolutions per minute).The mixing operation was then continued for a further 15 minutes whileincreasing the vacuum to 50 mbar. The D-calcium pantothenate-containinggranulate so prepared was then dried to a residual moisture content ofless than 2 wt. % in a vibratory fluidised-bed drier (Escher-Wyss,Linden, Germany) having a fluidised-bed surface area of 0.3 m² with abed temperature of 65° C. and a drying gas throughput of 270 Nm3/hour.Table 8 shows the average particle size distribution of the product.

TABLE 8 Designation Diameter Content [%] Fine dust <10 μm 1 Dust 10-50μm 2 Fines 50-125 μm 6 Mean particle size 200-400 μm 89  Oversizeparticles >400 μm 2

The content, measured as D-pantothenic acid, was determined as 32.6 wt.%. The product was almost dust-free. The apparent density was 650 kg/m³after drying.

By means of the process described herein, or by means of a differentspray, fluidised-bed, stirring or mixing process, the concentratecontaining D-calcium pantothenate or D-pantothenic acid can be sprayedonto other conventional organic or inorganic carriers or auxiliarysubstances, such as silicas, silicates, meals, brans, flours, starches,sugars, and granulated, optionally with the use of binders, gellingagents or other formulation auxiliaries.

Numerous modifications and variations of the present invention arepossible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described herein.

This application is based on German patent application serial No. 100 32349.9, filed on Jul. 4, 2000, the contents of which are incorporatedherein by reference.

What is claimed is:
 1. A process for preparing a feed additivecomposition, comprising: fermenting D-pantothenic acid, at least onesalt of D-pantothenic acid, or mixtures thereof in a fermentationliquor; separating a biomass from said fermentation liquor; drying thefermentation liquor; granulating said dried fermentation liquor toobtain particles having a size distribution of from 20 μm to 2000 μm;and compacting said fermentation liquor.
 2. The process of claim 1,further comprising adding at least one member selected from the groupconsisting of a hydroxide of an alkine earth metal, an oxide of analkaline earth metal, a hydroxide of an alkali metal, and an oxide of analkali metal to the fermentation liquor prior to drying the fermentationliquor.
 3. The process of claim 2, wherein the fermentation liquor isconcentrated prior to granulation.
 4. The process of claim 2, whereinthe oxide or hydroxide is added in a stoichiometric ratio of fomn 0.8 to1.2 based on the D-pantothenic acid.
 5. The process of claim 2, whereinthe oxide or hydroxide is added in a stoichiomefic ratio of from 0.95 to1.1 based on the D-pantothenic acid.
 6. The process of claim 1, furthercomprising adding the particles to animal feed.
 7. A process forpreparing a feed additive composition, comprising: fermentingD-pantothienic acid, at least one salt of D-pantothenic, acid, ormixtures thereof in a fermentation liquor; separating a biomass fromsaid fermentation liquor, drying the fermentation liquor wherein thedrying is performed by spray-drying the fermentation liquor; andgranulating said dried fermentation liquor to obtain particles having asize distributon of from 20 μm to 2000 μm.
 8. The process of claim 7,further comprising adding at least one member selected from the groupconsisting of a hydroxide of an alkaline earth metal, an oxide of analkaline metal, a hydroxide of an alkali metal, and an oxide of analkali metal to the fermentation liquor prior to drying the fermentationliquor.
 9. The process of claim 8, wherein the fermentation liquor isconcentrated prior to graulation.
 10. The process of claim 8, whereinthe oxide or hydroxide is added in a stoichiometric ratio of from 0.8 to1.2 based on the D-pantothenic acid.
 11. The process of claim 8, whereinthe oxide or hydroxide is added in a stoichiormatic ratio of from 0.95to 1,1 based on the D-pantothenic acid.
 12. The process of claim 7,further comprising adding the particles to animal feed.
 13. A processfor preparing a feed additive composition, comprising: fementingD-pantoeic acid, at least one salt of D-pantothenic acid, or mixtuesthereof in a fermentation liquor; separating a biomass from saidfermentation liquor, drying the fermentation liquor, and granulating thedried fermentation liquor to obtain partiles having a size distributionof from 20 μm to 2000 μm wherein the granulating is performed bybuild-up granulating the fermentation liquor.
 14. The process of claim13, further comprising adding at least one member selected from thegroup consisting of a hydroxide of an alkaline earth metal, an oxide ofan alkaline earth metal, a hydroxide of an akali metal, and an oxide ofan alkali metal to the fermentation liquor prior to drying thefermentation liquor.
 15. The process of claim 14, wherein thefermentation liquor is concentrated prior to granulation.
 16. Theprocess of claim 14, wherein the oxide or hydroxide is added in astoichiometric ratio of 0.8 to 1.2 based on the D-pantothenic acid. 17.The process of claim 14, wherein tie oxide or hydroxide is added in astoichiometric ratio of from 0.95 to 1.1 based on the D-pantothenicacid.
 18. The process of claim 13, farther comprising adding theparticles to animal feed.